1887

Abstract

Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation.

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2009-05-01
2024-12-01
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References

  1. Allison S. L., Stiasny K., Stadler K., Mandl C. W., Heinz F. X. 1999; Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J Virol 73:5605–5612
    [Google Scholar]
  2. Chambers T. J., Hahn C. S., Galler R., Rice C. M. 1990; Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649–688 [CrossRef]
    [Google Scholar]
  3. Elshuber S., Mandl C. W. 2005; Resuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virus. J Virol 79:11813–11823 [CrossRef]
    [Google Scholar]
  4. Elshuber S., Allison S. L., Heinz F. X., Mandl C. W. 2003; Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J Gen Virol 84:183–191 [CrossRef]
    [Google Scholar]
  5. Gazina E. V., Mackenzie J. M., Gorrell R. J., Anderson D. A. 2002; Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae . J Virol 76:11113–11122 [CrossRef]
    [Google Scholar]
  6. Guirakhoo F., Bolin R. A., Roehrig J. T. 1992; The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 191:921–931 [CrossRef]
    [Google Scholar]
  7. Hayes E. B., Komar N., Nasci R. S., Montgomery S. P., O'Leary D. R., Campbell G. L. 2005a; Epidemiology and transmission dynamics of West Nile virus disease. Emerg Infect Dis 11:1167–1173 [CrossRef]
    [Google Scholar]
  8. Hayes E. B., Sejvar J. J., Zaki S. R., Lanciotti R. S., Bode A. V., Campbell G. L. 2005b; Virology, pathology, and clinical manifestations of West Nile virus disease. Emerg Infect Dis 11:1174–1179 [CrossRef]
    [Google Scholar]
  9. Komar N., Panella N. A., Langevin S. A., Brault A. C., Amador M., Edwards E., Owen J. C. 2005; Avian hosts for West Nile virus in St. Tammany Parish. Louisiana: 2002 Am J Trop Med Hyg 73:1031–1037
    [Google Scholar]
  10. Konishi E., Mason P. W. 1993; Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol 67:1672–1675
    [Google Scholar]
  11. Li J., Bhuvanakantham R., Howe J., Ng M. L. 2006; The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes. J Gen Virol 87:613–622 [CrossRef]
    [Google Scholar]
  12. Li L., Lok S. M., Yu I. M., Zhang Y., Kuhn R. J., Chen J., Rossmann M. G. 2008; The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319:1830–1834 [CrossRef]
    [Google Scholar]
  13. Lin Y. J., Wu S. C. 2005; Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus. J Virol 79:8535–8544 [CrossRef]
    [Google Scholar]
  14. Lorenz I. C., Allison S. L., Heinz F. X., Helenius A. 2002; Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 76:5480–5491 [CrossRef]
    [Google Scholar]
  15. Mackenzie J. M., Westaway E. G. 2001; Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75:10787–10799 [CrossRef]
    [Google Scholar]
  16. Mackenzie J. M., Jones M. K., Westaway E. G. 1999; Markers for trans -Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. J Virol 73:9555–9567
    [Google Scholar]
  17. Ng M. L., Yeong F. M., Tan S. H. 1994; Cryosubstitution technique reveals new morphology of flavivirus-induced structures. J Virol Methods 49:305–314 [CrossRef]
    [Google Scholar]
  18. Op De Beeck A., Molenkamp R., Caron M., Ben Younes A., Bredenbeek P., Dubuisson J. 2003; Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope. J Virol 77:813–820 [CrossRef]
    [Google Scholar]
  19. Rust R. C., Landmann L., Gosert R., Tang B. L., Hong W. 2001; Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 75:9808–9818 [CrossRef]
    [Google Scholar]
  20. Sejvar J. J., Bode A. V., Marfin A. A., Campbell G. L., Ewing D., Mazowiecki M., Pavot P. V., Schmitt J., Pape J. other authors 2005; West Nile virus-associated flaccid paralysis. Emerg Infect Dis 11:1021–1027 [CrossRef]
    [Google Scholar]
  21. Stadler K., Allison S. L., Schalich J., Heinz F. X. 1997; Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71:8475–8481
    [Google Scholar]
  22. Wengler G., Wengler G. 1989; Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J Virol 63:2521–2526
    [Google Scholar]
  23. Yoshii K., Goto A., Kawakami K., Kariwa H., Takashima I. 2008; Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus. J Gen Virol 89:200–211 [CrossRef]
    [Google Scholar]
  24. Zhang Y., Corver J., Chipman P. R., Zhang W., Pletnev S. V., Sedlak D., Baker T. S., Strauss J. H., Kuhn R. J., Rossmann M. G. 2003; Structures of immature flavivirus particles. EMBO J 22:2604–2613 [CrossRef]
    [Google Scholar]
  25. Zhang Y., Kaufmann B., Chipman P. R., Kuhn R. J., Rossmann M. G. 2007; Structure of immature West Nile virus. J Virol 81:6141–6145 [CrossRef]
    [Google Scholar]
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